Curcusinol from the fruit of Carex baccans with antibacterial activity against multidrug-resistant strains.

ETHNOPHARMACOLOGICAL RELEVANCE: Carex baccans, known as Shan-Bai-Zi or Ye-Gao-Liang in China, is a traditional medicinal herb used by several ethnic groups in Yunnan Province. It is utilized for the treatment of wound infections, ulcers, and dysentery. However, there is currently a dearth of research reports on its antimicrobial potential. AIM OF THE STUDY: The substance basis of the antimicrobial activity of C. baccans will be unveiled, and the in vitro and in vivo antibacterial activities against multidrug-resistant bacteria of its major active compounds, as well as their preliminary mechanisms of action, will be investigated. MATERIALS AND METHODS: An antibacterial bioactivity-guided isolation method was used to isolate and identify the active compound curcusinol from C. baccans. UPLC-DAD-MS was employed for the quantitative analysis of curcusinol. The antibacterial activity, resistance profile, synergistic effects, anti-biofilm activity, and potential mechanisms of action of curcusinol against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and other multidrug-resistant bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) were investigated using various methods, including the broth microdilution method, scanning electron microscopy, time kill assay, multi-generational resistance induction assay, checkerboard synergy assay, anti-biofilm assay, and metabolomics. Furthermore, the therapeutic efficacy of curcusinol was assessed in vivo by establishing an animal skin wound infection model of MRSA. RESULTS: Curcusinol was isolated from the fruit of C. baccans, which accounts for 3.1% of the dry weight of the fruit. Curcusinol exhibited significant bactericidal and anti-biofilm activities against antibiotic-resistant Gram-positive bacteria in vitro. Furthermore, curcusinol acted as an antibiotic adjuvant to enhance the activity of various commonly used antibiotics against both Gram-positive and Gram-negative antibiotic-resistant bacteria without cytotoxicity to mammalian cells (A549 and RAW264.7) at 64 µM. Moreover, curcusinol affected arginine biosynthesis, cysteine and methionine metabolism, and alanine, aspartate, and glutamate metabolism significantly in MRSA cells under stress. Additionally, curcusinol effectively treated MRSA-infected mouse skin wounds and accelerated wound healing in vivo. CONCLUSIONS: The results of this study not only support the traditional uses of C. baccans but also demonstrate that its major active compound, curcusinol, is an effective plant-derived bactericidal agent and antibacterial adjuvant with potential applications in the treatment of skin infections.

Diterpenes of Pinus pinaster aiton with anti-inflammatory, analgesic, and antibacterial activities.

ETHNO-PHARMACOLOGICAL RELEVANCE: The P. pinaster species, known as 'Pino nigral or rodeno', is used in the treatment of colds, asthma, flu, and tuberculosis. AIM OF THE STUDY: This study determined the anti-inflammatory, analgesic, and antibacterial activities of the P. pinaster resin, identifying the compounds with higher biological activity. MATERIALS AND METHODS: A bio-guided isolation of the compounds of P. pinaster was carried out by selecting the most active extracts with anti-inflammatory and analgesic effects in the HBEC3-KT, MRC-5, and THP-1 cell lines. The antibacterial activity was determined against the S. aureus, S. pneumoniae, K. pneumoniae and P. aeruginosa strains. RESULTS: The following compounds were identified by NMR: dehydroabietic acid (1), ( + )-cis-abienol (2), pimaric acid (3), isopimaric acid (4), 7α-hydroxy-dehydroabietic acid (5), 7-oxo-dehydroabietic acid (6), 15-hydroxy-abietic acid (7), 7-oxo-15-hydroxy-dehydroabietic acid (8), 13-oxo-8 (14)-podocarpen-18-oic acid (9), and pinyunin A (10). Regarding their anti-inflammatory activity, all compounds inhibited NF-κB. Compound 9 was the most active (IC50 = 3.90-12.06 µM). Concerning the analgesic activity, all the compounds inhibited NK-1, yet compound 9 was the most active (IC50 = 0.28-0.33 µM). Finally, compounds 6 (MIC = 12.80-25.55 µM) and 9 (MIC = 9.80-24.31 µM) were the most promising antibacterial compounds in all strains. CONCLUSION: This study managed to identify, for the first time, six diterpenes from the resin of P. pinaster, with anti-inflammatory, analgesic, and antibacterial activity. Among the identified compounds, compound 9 was the most active, being considered a promising candidate as an antagonist of the tachykinin NK-1 receptor and as an analgesic agent against inflammation and neuropathic pain. It also had an antibacterial effect against Gram negative bacteria.

In vitro anti-bactrical activity and its preliminary mechanism of action of the non-medicinal parts of Sanguisorba officinalis L. against Helicobacter pylori infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Sanguisorba officinalis L. (S. officinalis L.), known as Di Yu (DY) in Traditional Chinese Medicine (TCM), are used to treat burns, vomiting of blood, asthma, intestinal infections, and dermatitis. It has been reported that the root of DY has a significant inhibitory effect on Helicobacter pylori (H. pylori). However, there is currently little research on the composition analysis and anti-H. pylori infection properties of the non-medicinal parts of DY, such as its stems, leaves, and flowers. AIM OF STUDY: The commonly used eradication therapies for H. pylori infection are antibiotic-based therapies. With the increasing antibiotic resistance of H. pylori, it is urgent to find effective alternative therapies. To find alternative therapies and increase the utilization of DY, this study aims to investigate the phytochemistry profile, in vitro anti-H. pylori activity, and preliminary antibacterial mechanism of the non-medicinal parts of DY. MATERIALS AND METHODS: The non-medicinal parts of DY extracts were obtained by using hot water reflux method. The chemical composition of these extracts was analyzed using colorimetric method, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). The in vitro anti-H. pylori activity was investigated using broth microdilution method, checkerboard dilution method, time-kill curve, time-inhibition curve, scanning electron microscopy, and transmission electron microscopy. Transcriptional sequencing technology was used to study the effect of DY stems and flowers on the gene expression of H. pylori and explore possible antibacterial mechanisms. RESULTS: The non-medicinal parts of DY contain abundant phytochemicals, such as total phenols and total flavonoids, and possess strong inhibitory and bactericidal activity against both standard and clinical strains of H. pylori in vitro. The MIC was 80-1280 µg/mL and the MBC was 80-2560 µg/mL, and the strength of the antibacterial effects was dependent on the concentration of phytochemicals (total polyphenols, gallic acid and ellagic acid). In addition, the combination of non-medicinal parts of DY with antibiotics, such as amoxicillin, metronidazole, levofloxacin, and clarithromycin, did not result in any antagonistic effects. All of them could disrupt the morphology, internal microscopic and cell wall structures of H. pylori thereby acting as an inhibitor. The mechanism of action was found to be the disruption of H. pylori morphology, internal microstructure, and cell wall. Transcriptomic analysis showed that the non-medicinal parts of DY significantly regulated the gene expression of H. pylori, especially the metabolic pathway. CONCLUSIONS: This study analyzed the chemical composition of the non-medicinal parts of DY and confirmed its inhibitory and bactericidal activities against H. pylori, both standard and clinical strains. Additional, the mechanism of inhibition involves disrupting the structure of H. pylori cells, altering gene expression, and interfering with bacterial metabolic pathways. This study provides a reference for further resource utilization and the development of H. pylori drugs using the non-medicinal parts of DY.

Prussian blue (PB) modified gold nanoparticles as a SERS-based sensing platform for capturing and detection of pyrazinoic acid (POA).

Pyrazinoic acid (POA) is a metabolite of the anti-tuberculosis drug pyrazinamide (PZA), and its detection can be used to assess the resistance of Mycobacterium tuberculosis in cultures, as only sensitive strains of the bacteria can metabolize PZA into POA. Prussian blue is a well-known metal-organic framework compound widely used in various sensing platforms such as electrochemical, photochemical, and magnetic sensors. In this study, we present a novel sensing platform based on Prussian blue-modified gold nanoparticles (AuNPs) designed to enhance the affinity of POA towards the sensing surface and to capture POA molecules from aqueous solutions. This SERS-based method allows for the selective enrichment of POA, which can be detected in both pure aqueous solution and in the presence of its pro-drug PZA. The limit of detection (LOD) for POA was estimated to be 1.08 µM in pure aqueous solution and 0.18 mM in the presence of PZA. Furthermore, the precision of the SERS method was verified by the relative standard deviation (RSD) of 3.34-12.02% for three parallel samples using different matrices, i.e. aqueous solution, spiked river water and spiked simulated saliva. The recoveries of the samples ranged from 92.65 to 118.51%. These all demonstrate the potential application of the proposed detection scheme in medical research.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding / Isolamento e caracterização de bactérias associadas ao trato intestinal do bicho-da-seda sob alimentação larval tratada com antibióticos

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Distribution and antibiogram of Vibrio species from hospital wastewater in Southwest, Nigeria.

Introduction: the continuous generation of wastewater and its release into the environment with little or no treatment remains a threat to the environment and public health. We examined the prevalence and antibiotic susceptibility profiles of Vibrio species isolated from untreated wastewater samples from Ondo State Specialist Hospital Okitipupa, Nigeria, as part of the global efforts to provide information for containing the spread of resistant infections. Methods: twelve hospital wastewater samples were collected aseptically and transported to the laboratory for analysis. The samples were processed on thiosulphate citrate bile salt sucrose agar and colonies typical of Vibrio species were selected for further identification. All isolates were confirmed by polymerase chain reaction (PCR) using Vibrio-specific primers and the PCR products were sequenced for species identification. The susceptibility profiles of the isolates were determined by the Kirby-Bauer disc diffusion method. Results: twenty-nine (58%) of 38 presumptive isolates were confirmed as Vibrio by PCR, while 23 (60.5%) isolates were screened up to species level by sequencing. Six different species following the trend: 26.1% V. fortis and V. algivorus, 17.4% V. cholerae, 13.0% V. panuliri, 8.7% V. stylophorae and V. parahaemolyticus were identified. The isolates were commonly resistant (73.9%-91.3%) to doxycycline, tetracycline, erythromycin and meropenem. The least resistance rate (17.4%) was observed against amikacin and cotrimoxazole. All isolates were multidrug-resistant, with multiple antibiotic resistance (MAR) indices exceeding the 0.2 recommended limit. Conclusion: this study has shown that untreated hospital wastewater is a reservoir for diverse strains of multiply resistant Vibrio species. Therefore, it is essential to adequately treat hospital wastewater to eliminate these emerging pollutants and set up a monitoring scheme to evaluate the treatment plants' effectiveness to reduce the pollutants' impact on the environment and the population.

Clinical and Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Infections in a Tertiary Hospital in China.

Purpose: Infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-KP) are an important public health problem. This study aimed to evaluate the clinical characteristics of patients with CR-KP. Methods: A retrospective cohort study was conducted of all patients with CR-KP infection. A total of 615 patients with CR-KP infection were identified and 135 patients who did not meet the eligibility criteria were excluded. Clinical characteristics, antimicrobial regimens, and patient outcomes were analyzed. Results: The overall mortality rate of CR-KP infections was 37.3% and the mortality rate in patients with bloodstream infections was 66.2%. Survival analysis revealed that there were statistically significant differences between patients with bloodstream infections and those with pulmonary and drainage fluid infections. Logistics regression analysis showed that hemopathy, age >60 years, solid tumors, diabetes, septic shock, acute kidney injury, and stroke were independent predictors of 30-day mortality rate. The chi-square test showed that treatment with a combination of carbapenems, tigecycline, and polymyxin B was superior to treatment with carbapenems with polymyxin B, without tigecycline. Conclusions: CR-KP infections, especially bloodstream infections, have a high mortality rate. The outcome is strongly dependent on patients' clinical conditions. Antimicrobial regimens combining carbapenems, tigecycline, and polymyxin B might be a better choice.

Use and Quality of Blood Cultures for the Diagnosis of Bloodstream Infections: A Cross-Sectional Study in the Ho Teaching Hospital, Ghana, 2019-2021.

Blood Culture and Drug Susceptibility Testing (CDST) remains vital for the diagnosis and management of bloodstream infections (BSIs). While the Ghana National Standard Treatment Guidelines require CDST to be performed in each case of suspected or clinically diagnosed BSI, these are poorly adhered to in the Ho Teaching Hospital (HTH). This study used secondary medical and laboratory records to describe blood CDST requests by clinicians and the quality of CDST processes for the diagnosis of BSI among patients admitted to HTH from 2019 to 2021. Of 4278 patients, 33% were infants. Pneumonia and neonatal sepsis cases were 40% and 22%, respectively. Only 8% (351/4278) had blood CDST requested. Of 94% (329/351) blood CDST processed and reported, only 7% (22/329) were culture-positive, with likely contaminants being recovered from 16% (52/329) of the specimens. The duration from admission to request was 2 days (IQR: 0-5), and Further qualitative studies must be conducted to understand the reasons for low blood CDST utilisation among clinicians and the patient outcomes. Targeted interventions are required to enhance the utilisation of blood CDST by clinicians and the quality of laboratory processes.

Phenotypic and genotypic drug susceptibility patterns of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Central and Southern Ethiopia.

INTRODUCTION: The persistence of tuberculosis (TB) infection in some patients after treatment has highlighted the importance of drug susceptibility testing (DST). This study aimed to determine the drug susceptibility patterns of Mycobacterium tuberculosis (M. tuberculosis) isolates from pulmonary TB (PTB) patients in Central and Southern Ethiopia. METHODS: A health institution-based cross-sectional study was conducted between July 2021 and April 2022. Sputum samples were collected from newly diagnosed smear microscopy and/or Xpert MTB/RIF-positive PTB patients. The samples were processed and cultivated in Lowenstein-Jensen (LJ) pyruvate and glycerol medium. M. tuberculosis isolates were identified using polymerase chain reaction (PCR) based region of difference 9 (RD9) deletion typing. Phenotypic DST patterns of the isolates were characterized using the BACTEC MGIT™ 960 instrument with SIRE kit. Isoniazid (INH) and Rifampicin (RIF) resistant M. tuberculosis isolates were identified using the GenoType® MTBDRplus assay. RESULTS: Sputum samples were collected from 350 PTB patients, 315 (90%) of which were culture-positive, and phenotypic and genotypic DST were determined for 266 and 261 isolates, respectively. Due to invalid results and missing data, 6% (16/266) of the isolates were excluded, while 94% (250/266) were included in the paired analysis. According to the findings, 14.4% (36/250) of the isolates tested positive for resistance to at least one anti-TB drug. Gene mutations were observed only in the rpoB and katG gene loci, indicating RIF and high-level INH resistance. The GenoType® MTBDRplus assay has a sensitivity of 42% and a specificity of 100% in detecting INH-resistant M. tuberculosis isolates, with a kappa value of 0.56 (95%CI: 0.36-0.76) compared to the BACTEC MGIT™ DST. The overall discordance between the two methods was 5.6% (14/250) for INH alone and 0% for RIF resistance and MDR-TB (resistance to both INH and RIF) detection. CONCLUSION: This study reveals a higher prevalence of phenotypic and genotypic discordant INH-resistant M. tuberculosis isolates in the study area. The use of whole-genome sequencing (WGS) is essential for gaining a comprehensive understanding of these discrepancies within INH-resistant M. tuberculosis strains.

Oral candidiasis in patients with kidney transplantation in Iran: prevalence and antifungal susceptibility pattern.

Aim: This study aimed to identify Candida species recovered from the oral cavity of patients with kidney transplantation. Materials & methods: Two swabs were taken from the oral cavities of 40 patients before and after transplantation, cultured on Sabouraud dextrose agar, and yeasts identified. Antifungal drug susceptibility testing was performed with fluconazole and itraconazole. Results: Candida glabrata was the most frequently isolated species in patients, followed by Candida albicans and Rhodotorula. C. glabrata isolates from patients before transplantation were resistant to fluconazole, whereas C. albicans was fluconazole-resistant both before and after transplantation. Conclusion: The importance of non-albicans Candida species in the oral cavity of patients sheds light on performing antifungal tests for achieving the best outcome to prevent therapeutic failure.

[Effects of rapid drug sensitivity testing for multidrug-resistant bacteria on the prognosis of patients with severe intra-abdominal infection].

Objective: To examine the clinical value of rapid detection of drug-resistant bacteria by immunochromatography and the effects of rapid detection on the prognosis of patients with severe intra-abdominal infection complicated by carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infection. Methods: This was a retrospective cohort study. We analyzed clinical data of 73 patients with severe abdominal infections with sepsis or septic shock complicated by CRE bloodstream infection admitted to the general surgery department of Jinling Hospital between February 2022 and February 2023. Patients were divided into a colloidal gold immunochromatographic assay (GICA) group (17 patients) and conventional testing group (56 patients) based on whether a GICA for CRE had been performed on the patients' first blood culture sample during the diagnosis and treatment process. There were no statistically significant differences between the GICA and conventional testing groups in age ([55.9±17.3] vs. [47.6±16.4] years), sex ([16 men vs. one woman ] vs. [41 men vs. 15 women]), median Charlson comorbidity index (3.0[2.0,4.0] vs. 3.0[2.0, 4.8]), septic shock (10 vs. 39), or acute kidney injury (8 vs. 40) (all P>0.05). Both groups routinely underwent traditional bacterial identification and drug susceptibility testing. Additionally, patients in the GICA group were tested directly for positive blood cultures using a GICA carbapenemase test kit. The main outcomes were mortality rates on Days 28 and 90 after the first identification of CRE bloodstream infection in both groups. We also compared the microbial clearance rate, duration of hospitalization and intensive care unit stay, and time from onset of CRE bloodstream infection to initiation of targeted and appropriate antibiotics between the two groups. Results: The rate of microbial clearance of bloodstream infection was significantly greater in the GICA group than in the conventional testing group (15/17 vs. 34/56 [60.7%], χ2=4.476, P=0.034), whereas the 28-day mortality tended to be lower in the GICA than conventional testing group [5/17 vs. 44.6% [25/56], χ2=1.250, P=0.264). The 90-day mortality (8/17 vs. 53.6% [30/56], χ2=0.222, P=0.638), median duration of hospitalization (37.0 [18.0, 46.5] days vs. 45.5 [32.2, 64.8] days, Z=-1.867, P=0.062), and median duration of intensive care unit stay (18.0 [6.5, 35.0] days vs. 32.0 [5.0, 51.8] days, Z=-1.251, P=0.209). The median time between the onset of bloodstream infection and administration of antibiotics was 49.0 (38.0, 69.0) hours in the GICA group, which is significantly shorter than the 163.0 (111.8, 190.0) hours in the conventional testing group (Z=-5.731, P<0.001). The median time between the onset of bloodstream infection and administration of appropriate antibiotics was 40.0 (34.0, 80.0) hours in the GICA group, which is shorter than in the conventional testing group (68.0 [38.2, 118.8]) hours; however, this difference is not statistically significant (Z=-1.686, P=0.093). Conclusions: GICA can provide information on carbapenemase- producing pathogens faster than traditional drug sensitivity testing, enabling early administration of the optimal antibiotics. The strategy of 'carbapenemase detection first' for managing bacterial infection has the potential to improve prognosis of patients and reduce mortality rate.

Presence of T3SS (exoS, exoT, exoU and exoY), susceptibility pattern and MIC of MDR-Pseudomonas aeruginosa from burn wounds.

INTRODUCTION: The emergence of resistance is a major public health and clinical issue, particularly in pathogens causing nosocomial infections. Recently, there is the emergence of Pseudomonas aeruginosa resistance to different broad-spectrum antibiotics. METHODOLOGY: The current study was designed to find out the prevalence of multi-drug resistant (MDR) P. aeruginosa in burn patients, the antibiotic susceptibility pattern of MDR Pseudomonas, and to determine the Minimum Inhibitory Concentration (MIC) of the effective antimicrobials. The assessment of virulence genes (exoT, exoS, exoY and exoU) was also achieved through PCR. In the current study wound swabs were collected from 160 burn patients from two burn units (MTI-Govt. Lady Reading Hospital and MTI-Khyber Teaching Hospital). RESULTS: Out of these 160 samples, 26 samples (16.25%) were positive for P. aeruginosa. Per patients, one isolate was included in the current study. Antibiotic susceptibility pattern showed all P. aeruginosa isolates were 100% resistant to amoxicillin-clavulanic acid, 84.62% resistance to Cefepime, and Ceftazidime, and 76.92% resistance to Amikacin, Aztreonam, and Ciprofloxacin. Whereas the lowest resistance was observed to Imipenem and Piperacillin-Tazobactam (53.85%), Colistin Sulfate (23.08%), and Polymyxin-B (15.38%). Regarding the prevalence of MDR, 22 (84.61%) isolates out of 26 were found to be MDR-P. aeruginosa. For MDR-P. aeruginosa, the MIC range was 1-2 µg/mL against Polymyxin-B, 2-8 µg/mL against Colistin sulfate, 16-1024 µg/mL against Imipenem and 128-1024 µg/mL against Piperacillin-Tazobactam. 100% of the isolates carried exoT, 88.46% carried exoY, and 57.69% and 38.46% carried exoU and exoS, respectively. CONCLUSIONS: These findings further emphasize the need for antibiotic discipline and to follow the recommended hospital antibiotic policy to prevent the proliferation of MDR strains of P. aeruginosa in the community.

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

INTRODUCTION: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. METHODOLOGY: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. RESULTS: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. CONCLUSIONS: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

Bacteriological Profile of Surgical Site Infection Following Gastrointestinal Surgery and Their Antibiogram.

BACKGROUND: Surgical site infection is one of the common complication following abdominal surgery. It causes great morbidity and mortality, further increasing prevalence of multidrug resistant bacteria have made its management very challenging. The current study aims to identify causative agent responsible for surgical site infection and their antibiotic resistance patterns. METHODS: This study was conducted among patients developing surgical site infection following gastrointestinal surgery in Tribhuvan university teaching hospital over a period of one year. The samples were collected and processed according to standard methods. The bacterial pathogens with their antimicrobial susceptibility were determined and resistant pattern like methicillin resistant Staphylococcus aureus and extended spectrum beta lactamase were further detected. RESULTS: A total of 832 patients had under gone gastrointestinal surgery during the study period. Among them, 162 cases (19.5%) developed surgical site infection and 125 cases showed growth in culture. A total of 160 aerobic bacteria were isolated; Escherichia coli (29.9%) was the commonest organism with 40.8% being extended spectrum beta lactamase producer and 47.4% of Staphylococcus aureus were methicillin resistant. About 75.9% (85/112) of gram negative bacteria and 60.4% (29/48) gram positive bacteria were multi drug resistant. CONCLUSIONS: The burden of multi drug resistant bacteria causing surgical site infection is high which needs to be addressed timely. Good surveillance of bacterial antibiogram and rational antimicrobial use is necessary to reduce emergence and spread of resistant bacteria.

Recomendaciones del Comité Español del Antibiograma (COESANT) para la realización de los Informes de Sensibilidad Antibiótica Acumulada / Spanish Antibiogram Committee (COESANT) recommendations for cumulative antibiogram reports

El Comité Español del Antibiograma (COESANT) presenta en este documento una serie de recomendaciones cuya finalidad es unificar la forma en la que los Servicios y Unidades de Microbiología Clínica españoles realizan los informes de sensibilidad acumulada de las bacterias, aisladas en muestras clínicas, frente a los antimicrobianos. Las recomendaciones se fundamentan en las recogidas en el Procedimiento de Microbiología Clínica n° 51, «Preparación de informes acumulados de sensibilidad a los antimicrobianos» de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), publicado en 2014, y recoge las modificaciones en las definiciones de las interpretaciones de las categorías clínicas publicadas en el año 2019 por el European Committee on Antimicrobial Susceptibility Testing (EUCAST). Su objetivo final es establecer una forma homogénea de elaborar estos resúmenes para poder comparar resultados de diferentes centros o sumar su información y así realizar una adecuada vigilancia local o incluso nacional de la evolución de la sensibilidad a los antimicrobianos.(AU)

The Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) presents in this document a series of recommendations intending to unify how cumulative antibiogram reports must be made in Clinical Microbiology Spanish laboratories. This article is based on the information included in the Clinical Microbiology Procedure No. 51, «Preparation of cumulative reports on antimicrobial susceptibility» of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), published in 2014. The recommendations also include the modifications in the definition of clinical interpretive categories recently published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2019. Its final objective is to establish a homogeneous way of preparing these summaries to compare results from different centers or aggregate the information from these in order to carry out an adequate local or even national surveillance regarding the evolution of antimicrobial susceptibility.(AU)

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes.

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked. IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Evaluation of the liquid colony for identification and antimicrobial susceptibility testing directly from positive blood cultures.

BACKGROUND: Sepsis represents a time-sensitive disease requiring early therapeutical intervention to avoid adverse patient outcomes. Rapid microbiological diagnosis is essential to investigate sepsis aetiological agents. The FAST™ system (Qvella, ON, Canada) provides a concentrated microbial suspension, known as a Liquid Colony™ (LC), directly from positive blood samples (PBCs) in 30-40 min to perform rapid identification (ID) and antimicrobial susceptibility testing (AST). METHODS: Qvella's FAST™ System and FAST PBC Prep cartridges were tested on PBCs from the Policlinico Hospital of Catania during a six-month study. Two millilitres of PBC were converted into an LC for rapid ID and AST using Bruker Biotyper Sirius MALDI and BD Phoenix systems. Standard of care (SOC) methods were used as a reference, requiring 48-72 h. Agreement between the innovative technology and the standard method was calculated. RESULTS: FAST System processing was performed on 100 monomicrobial PBCs. Median turnaround times from blood cultures flagging positive to ID and AST completion were 2 and 26 h respectively. Therefore, the LC procedure was 24 h faster than the median turnaround times for SOC methods. 100% ID identification concordance was observed across 48 Gram-negative bacteria, 42 Gram-positive bacteria and 11 yeast for the genus level. 78% of Gram-negative and 95% of Gram-positive bacteria were resistant to ≥ 2 antimicrobial agents, including 45% (15/33) carbapenem-resistant enteric Gram-negative bacteria and 90% (28/31) oxacillin-resistant staphylococci. An AST essential agreement of 100% was observed due to the absence of MIC discrepancies > 1-fold dilution. Categorical errors were not observed due to the absence of MIC categorization discordances. Yeast AST was not performed. CONCLUSIONS: The Qvella FAST System produces an LC that reliably reflects the MALDI spectra and phenotypic antimicrobial susceptibility profile of microbial cells growing in the blood culture. Timely processing of PBCs with the Qvella FAST System enables sepsis diagnostic confirmation 1 day sooner than the standard methods. In line with these results, it is vital now to focus attention on establishing best practices for incorporating this strategic tool into the clinical microbiology laboratory workflow.

Determining cost and placement decisions for moderate complexity NAATs for tuberculosis drug susceptibility testing.

BACKGROUND: Access to drug resistant testing for tuberculosis (TB) remains a challenge in high burden countries. Recently, the World Health Organization approved the use of several moderate complexity automated nucleic acid amplification tests (MC-NAAT) that have performance profiles suitable for placement in a range of TB laboratory tiers to improve drug susceptibility tests (DST) coverage. METHODS: We conducted cost analysis of two MC-NAATs with different testing throughput: Lower Throughput (LT, < 24 tests per run) and Higher Throughput (HT, upto 90+ tests per run) for placement in a hypothetical laboratory in a resource limited setting. We used per-test cost as the main indicator to assess 1) drivers of cost by resource types and 2) optimized levels of annual testing volumes for the respective MC-NAATs. RESULTS: The base-case per test cost of $18.52 (range: $13.79 - $40.70) for LT test and $15.37 (range: $9.61 - $37.40) for HT test. Per test cost estimates were most sensitive to the number of testing days per week, followed by equipment costs and TB-specific workloads. In general, HT NAATs were cheaper at all testing volume levels, but at lower testing volumes (less than 2,000 per year) LT tests can be cheaper if the durability of the testing system is markedly better and/or procured equipment costs are lower than that of HT NAAT. CONCLUSION: Assuming equivalent performance and infrastructural needs, placement strategies for MC-NAATs need to be prioritized by laboratory system's operational factors, testing demands, and costs.

Synthesis, characterisation and antibacterial activity of novel Ga(III) polypyridyl catecholate complexes.

Ga(III) polypyridyl catecholate complexes of type [Ga(bipy)2(O,O)](NO3) or [Ga(phen)2(O,O)](NO3) respectively were readily synthesised on reaction of Ga(NO3)3 in methanol with 1 equivalent of catecholate ligand (2,3-DHBA, 3,4-DHBA, 2,3,4-THBA or CafA) and then 2 equivalents of either bipy or phen. The complexes were characterised in full including by X-ray crystallography, which established that the catecholate ligands coordinate the Ga(III) centres in a bidentate manner via the two deprotonated hydroxy groups. All Ga(III) complexes exhibited good in vitro antibacterial activity against the Gram-negative pathogenic bacteria Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. The complexes were inactive against the Gram-positive pathogenic bacteria Staphylococcus aureus including against a methicillin-resistant Staphylococcus aureus strain (MRSA). [Ga(bipy)2(2,3-DHBA-2H)](NO3)·1.5H2O (1) was shown to be non toxic in vivo in larvae of Galleria mellonella at doses up to 2000 µg mL-1 and to offer protection at doses of 100 and 250 µg mL-1 at 48 and 96 h to larvae infected with P. aeruginosa.